GPIIb/IIIa platelet receptors assay

ABSTRACT

The present invention relates to a method of determining, on a test sample, the occupation of the cellular GPIIb/IIIa receptor by an antagonist of fibrinogen, characterized in that the number of receptors occupied is determined in the said sample with the aid of an antibody MAb1 which is a competitor for the antagonist, and the total number of occupied or unoccupied receptors is determined with the aid of a so-called noncompetitor antibody MAb2 specific for the occupied or unoccupied receptor and the rate of occupation of the GPIIb/IIIa receptors in the sample is deduced therefrom.

This application is a national stage filing under 35 USC 371 fromPCT/FR98/01135, filed Jun. 4, 1998.

The invention relates to a method of analysing the platelet GPIIb/IIIareceptors occupied by an anti-platelet aggregation compound or byfibrinogen and the receptors which have remained in the free state.

The platelets play a key role in the equilibrium of the haemostaticbalance. Activating signals can cause, at their level, bothmorphological and biochemical modifications together with variations inthe levels of expression of surface glycoproteins. These glycoproteinsare, for most ligand receptors, involved in adhesion (eg. the receptorfor the von Willebrand factor: the molecule GPIb), in aggregation (eg.the receptor for fibrinogen: the molecule GPIIb/IIIa) whereas othersindicate the state of platelet activation (eg. the molecule GMP 140 orP-selectin).

The activation of platelets and the aggregation resulting therefrom arephysiological phenomena which, after exceeding a threshold, areassociated with various pathological conditions such as arterialthrombotic accidents. This explains the reason for the interest whichthe pharmaceutical industry has for developing new anti-aggregationtherapeutic molecules.

The preferred target in the new therapies developed is the GPIIb/IIIareceptor which is specifically expressed on the platelets. Under theeffect of an activation of the platelets, this receptor binds, with avery high affinity, fibrinogen and other adhesion proteins, which causesaggregation of the platelets with each other.

The GPIIb/IIIa receptor belongs to the group comprising the integrins,which is composed of various molecules involved in the phenomena of celladhesion. The integrins are a/p heterodimers and are classified intoseveral families according to their β-subunit. About twenty integrinsare currently known, of which 9 bind to their ligand via the amino acidsequence R—G—D.

GPIIb/IIIa (αIIβ3) is a member of the family of β3 integrins. It has thesame β subunit (but a different α subunit) as the vitronectin receptor(αvβ3) present especially on the platelets and, in a large quantity, onthe endothelial cells. The IIb and IIIa subunits are classifiedrespectively into the CD 41 and CD 61 groups according to theinternational nomenclature.

The anti-platelet GPIIb-IIIa anti-aggregation agents belong to twodifferent classes: a) the monoclonal antibodies which are antagonists offibrinogen, including C7E3Fab of the humanized 7E3 monoclonal antibodydirected against an epitope of CD 61 and marketed under the name a 7E3monoclonal antibody solid under the tradename REOPRO®, b) the cyclicpeptides, of the integrelin type, which possess the sequence RGD or KGD,close to the RGD sequence (Arg—Gly—Asp) common to the platelet receptorligands, and the peptidomimetics, of the lamifiban or tirofiban type.These currently developed substances are only active by the parenteralroute. Other molecules which can be administered by the oral route arebeing developed. These new molecules may, however, induce side effectswhich result in the patients treated in haemorrhagic effects at thepoints of access or of internal location. For these new molecules which,by their nature, remain in circulation for a long time, there iscurrently no antidote. It is therefore important, in the context of ananti-platelet therapy, to be able not only to adapt the therapeutic doseof the agent administered, but also to rapidly have available, duringthe monitoring of the treatment, information on the number of GPIIb/IIIareceptors occupied by the antagonist, so as to be able to control thehaemorrhagic complications while preserving a sufficient quantity ofbound antagonist in order to obtain an anti-aggregation effect.

Accordingly, the present invention relates to a method of determining,on a test sample, the occupation of the GPIIb/IIIa receptor, by anantagonist of fibrinogen, characterized in that the number of receptorsoccupied is determined with the aid of an antibody [MAb1] which is acompetitor for the antagonist, and the total number of occupied orunoccupied receptors is determined with the aid of a so-callednoncompetitor antibody [MAb2] specific for the occupied or unoccupiedreceptor and the occupation of the GPIIb/IIIa receptors in the sample isdeduced therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the determination of the saturating concentration of a 7E3monoclonal antibody sold under the tradename ReoPro® on a platelet atthe basal state and after activation with ADP.

FIG. 2 shows that when the a 7E3 monoclonal antibody sold under thetradename ReoPro® preincubation concentration is varied in adose/response experiment with protocol 3, the binding of P18 isinversely proportional to that of the anti-aggregation agent.

FIG. 3 is a saturation study on platelets for purified mabs CD 61 P18and PM 6/13.

FIG. 4 shows an in vitro model on whole blood with a 7E3 monoclonalantibody sold under the tradename ReoPro® as an anti-aggregation agentusing PM6/13 as Mab2 and P18 as Mab1.

FIG. 5 shows an in vitro model on whole blood with RGD peptide as ananti-aggregation agent using PM6/13 as Mab2 and P18 as Mab1.

FIG. 6 shows the number of free receptors after the injection relativeto the total number of receptors—has been inserted on page 3, betweenlines 9 and 10.

Occupation is understood to mean both the quantification, in absolutenumerical terms, of the platelet receptors for fibrinogen GPIIb/IIIaoccupied by an anti-aggregation agent compared with the total number ofreceptors in a blood sample, and a ratio whose variation may be studiedover time.

It is also possible to determine the total number of receptors beforeany medication without having to repeat this determination in thecontext of the monitoring of the treatment.

Likewise, when it is specified that the number of receptors isdetermined, this may only be a semiquantitative determination.

Of course, when it is desired to precisely measure the number ofreceptors, it is necessary to provide calibration series withcalibration curves.

The anti-GPIIb/IIIa antibodies used in the context of the presentinvention are preferably directed against an epitope of the GPIIIa(CD61)subunit.

They are more particularly the antibodies SZ 21 and P2 which aremarketed by the company Immunotech and the antibody PM 6/13 which ismarketed by the company Cymbus.

They may also be the monoclonal antibodies P18 deposited in the BelgianCoordinated Collection of Microorganisms (BCCM) under the number LMBP1662CB on Jun. 5, 1997 (this deposit is only accessible to experts, inaccordance with legal provisions).

The antibody, PM 6/13 is available from Cymbus Bioservices Limited (UK),it is an anti-CD 61 of the IgG1 SZ 21 type available from Immunotech(France) and an anti-CD 61 of the IgG1 type; as for P2 which isavailable from the same company, it is an anti-CD 41 IgG1.

The antibody pairs used will depend mainly on the type ofanti-aggregation agent used. Indeed, a 7E3 monoclonal antibody soldunder the tradename REOPRO® and peptides of the RGD type areanti-aggregation agents which bind to different sites of GPIIIa.

A 7E3 monoclonal antibody sold under the tradename REOPRO® has a siteclose to that for fibrinogen and inhibits its binding by sterichindrance; on the contrary, the RGD peptides have the same site asfibrinogen, in direct competition for the RGD sequence.

Because of this, the most suitable product for the determination of thereceptors occupied by a 7E3 monoclonal antibody sold under the tradenameREOPRO® is the pair consisting of the competitor antibody P2 fromImmunotech and the noncompetitor antibody PM 6/13 from Cymbus, whereasin the case of the RGD peptides, the competitor antibody is the antibodyPM 6/13 from Cymbus and the noncompetitor antibody is the antibody SZ 21from Immunotech. Indeed, the antibody PM 6/13 is a noncompetitorantibody when the anti-aggregation agent is a 7E3 monoclonal antibodysold under the tradename REOPRO®, but becomes a competitor antibody ifit is an RGD peptide.

The determination using the preceding mono-clonal antibodies ispreferably a determination by quantitative cytometry, but other methodsinspired in particular by Elisa or another type of method may beconsidered.

Preferably, the determination is carried out by revealing specificantibodies with the aid of antibodies labelled with a fluorochromeusing, in particular, the indirect immunofluorescence method which maybe combined with a quantitative method.

The so-called cytometric method, as well as the indirect quantitativeimmunofluorescence method (so-called IQIF method) are known and will notbe described again in detail (P. Poncelet et al., 1985, J. Immunol.Methods 85, 65-74).

The sample used is preferably a biological sample, especially a bloodsample, a sample of whole blood collected over sodium citrate or citratetheophilline adenosine dipyrridanol (CTAD) (inhibitor of the plateletfunctions), or alternatively a fraction rich in platelets, a plasma richin platelets (PRP) or bound platelets for example.

Although specific antibody pairs have been previously mentioned it ispossible to use other anti-bodies which may be selected in particularfrom the lists provided in the book entitled “Leucocyte Typing V, WhiteCell Differentiation Antigens; Proceedings of the Fifth InternationalWorkshop and Conference held in Boston, USA, Nov. 3-7, 1993, volume 2,Oxford University Press, 1995”, since for the anti-GPIIb/IIIa competitorantibodies, there is selected an antibody directed against an epitope ofthe GPIIb/IIIa complex whose rate of binding thereto drops by at least50% and preferably 75% in the presence of an anti-platelet aggregationagent used at saturating concentration, but whose binding should notinhibit that of the anti-aggregation agent to the platelets.

As regards the noncompetitor anti-GPIIb/IIIa antibody, it will be anantibody directed against an epitope of the GPIIb/IIIa pair whose rateof binding thereto does not vary more than 15% in the presence and inthe absence of an anti-platelet aggregation agent.

The present invention also relates to an assay kit intended for carryingout the preceding method, characterized in that it comprises at least:

one monoclonal antibody for the GPIIb/IIIa receptor which is acompetitor for a fibrinogen antagonist, and

one monoclonal antibody specific for the occupied or unoccupiedGPIIb/IIIa receptor.

The principle of the quantification of surface antigens such as theGPIIb/IIIa receptors can be briefly recalled here.

The cellular antigenic sites to be quantified are labelled in indirectimmunofluorescence, with binding of an mAb, preferably of the IgG typeand in particular from mice, specific for the antigen to be studied,revealed by an anti-mouse IgG polyclonal conjugated with a fluorochrome.The intensity of fluorescence obtained in flow cytometry is an arbitraryunit which is, however, closely linked to the cellular level ofexpression of the antigen. Standardization of the cytometric data isprovided by the introduction, during each immunolabelling, of acalibrating substance treated in parallel with the sample tubes. Thiscalibrating substance consists of latex beads coated with increasing anddefined quantities of IgG-type mAb. Analysis of this calibratingsubstance allows the preparation of a calibration series linking theintensities of fluorescence to the absolute number of antigens per cell.

The examples below make it possible to demonstrate the characteristicsand advantages of the present invention.

The test provided is carried out on a sample of whole blood collectedover sodium citrate (or CTAD). In the CYTOQUANT Gp model, the labellingtechnique is performed directly on whole blood and does not require awashing step. For this test, three parameters are studied per sample (3tubes): the blood is placed in contact with a negative-control mAb(nonspecific labelling), an mAb Mab1 and an mAB MAb2. The calibrationseries is added to one tube during the second labelling step, in thepresence of the fluorescent revealing Ab. The intensity of fluorescencedue to the immunolabelling is then read on a cytometer. This intensityof fluorescence is proportional to the number of specific mAb bound tothe platelets. Conversion of the intensity of fluorescence into absolutenumber of mAB bound per platelet is carried out after construction ofthe calibration straight line.

The results are expressed in the following manner:

total number of molecules of GPIIb/IIIa per platelet (MAb2 value)

number of GPIIb/IIIa molecules occupied by the anti-aggregation molecule(MAb2-MAb1 difference).

Thus, two parameters may be studied at the same time.

EXAMPLE 1

During the feasibility phase, various mAbs specific for GPIIb/IIIa weretested on platelets in the presence and in the absence of a 7E3monoclonal antibody sold under the tradename REOPRO®.

Determination of the saturating concentration of ReoPro on a platelet atthe basal state and after activation with ADP

Protocol 1

The indirect immunofluorescence test by replacing the specific mAb witha 7E3 monoclonal antibody sold under the tradename REOPRO® at differentdilutions.

1—Materials

a 7E3 monoclonal antibody sold under the tradename REOPRO® 2 mg/ml,dilutions in 0.1% PBS-BSA from 50 μg/ml→0.75 μg/ml

ADP Stago 0.2 μM

DDAF

2—Method

Mix vol/vol PRP/dilutions of a 7E3 monoclonal antibody sold under thetradename REOPRO ® (mix 1) PRP/PBS buffer (mix 2) Incubation 15 min RTMix vol/vol mixture 1/PBS mixture 1/ADP 0.2 μM mixture 2/PBS mixture2/ADP 0.2 μM

Incubation 5 min RT

To each of the mixtures, add 4 ml PBS BA. Centrifugation 3000 rpm 10min.

Remove the supernatant.

Pellet+100 μl DDAF (sheep anti-mouse-FITC reagent) Incubation 15 min RT

+2 ml PBS→FCM analysis N. B. There are as many mixture 1 tubes as ReoProdilutions.

a 7E3 monoclonal antibody sold under the tradename REOPRO® is a chimericF(ab)-type Ab with an immunoreactive mouse Fv portion combined with aconstant human IgG portion. The second reagent, an FITC-conjugatedanti-mouse Ig sheep polyclonal, binds weakly to the ReoPro molecule butsufficiently to obtain an analysable signal. The saturatingconcentration is from 1 μg/ml final concentration. Platelet activationwith ADP does not modify the binding of a 7E3 monoclonal antibody soldunder the tradename REOPRO®.

The results of the test are presented in FIG. 1.

Identification of mabs which are, on the one hand, complementary and, onthe other hand, competitors for the binding of ReoPro

Protocol 2

1—Materials

a 7E3 monoclonal antibody sold under the tradename REOPRO® 2 mg/mldiluted to 10 μg/ml in 0.1% PBS BSA

MAbs CD41 and CD61 (anti-GPIIb/IIIa and anti-GPIIIa) at 10 μg/ml.

DDAF (sheep anti-mouse Ig-FITC).

2—Method

Mix vol/vol PRP/a 7E3 monoclonal antibody sold under the tradenameREOPRO ® 10 μg/ml PRP/PBS BSA 0.1%

Incubation 5 min RT

Washing of the 2 mixtures (+4 ml PBS BSA 0.1%, centrifugation 3000 rpm10 min, remove the supernatant).

Pellet+PBS BSA (vol=initial vol of PRP)

20 μl of each mixture+20 μl of each MAb to be tested.

Incubation 10 min RT

+100 μl DDAF 1/100

Incubation 10 min RT

+2 ml PBS BSA 0.1% →FCM analysis

We tested 9 mAbs specific for GPIIb/IIIa (CD41 and CD61) (see Table 1).

TABLE 1 RGD PEPTIDE B14 TYPE % INHIBITION TYPE % INHIBITION P18(BIOCYTEX) Stable 3 Competitor 98 PM6/13 (CYMBUS) Competitor 83 Stable 7SZ21 (IMMUNOTECH) Stable 0 Stable +/− 14 P2 (IMMUNOTECH) Competitor 76Competitor 99 M148 (CYMBUS) Stable +/− 12 Stable 1 1PLA SD5 (BIOCYTEX)Stable 0 Competitor 100 1PLA 4F8 (BIOCYTEX) Competitor 79 Stable 0 P1(INSERM) Stable +/− 26 Stable +/− 11 P6 (INSERM) Stable 6 Stable 0 Testcarried out on whole blood in the absence of a drug (100%), and in thepresence of a saturating concentration of anti-aggregation agentinhibiting the binding of fibrinogen.

We compared the quantitative values obtained on platelets preincubatedin the presence of ReoPro (10 μg/ml final) or of PBS. We chose the mAbPM6/13 whose expression does not vary by more than 10% in the presenceand in the absence of a 7E3 monoclonal antibody sold under the tradenameREOPRO® and the mAb P18 whose expression drops by more than 95% in thepresence of a 7E3 monoclonal antibody sold under the tradename REOPRO®used at saturating concentration.

Table 1 shows the results obtained with the same antibodies tested onwhole blood.

The advantage of using the PM16/13-P18 pair lies in the comparableresponse observed on a platelet for the 2 mAbs, in the absence ofanti-aggregation agent.

When the a 7E3 monoclonal antibody sold under the tradename REOPRO®preincubation concentration is varied, in a dose/response experimentwith protocol 3, the binding of P18 is inversely proportional to that ofthe anti-aggregation agent as indicated in FIG. 2.

Conclusion

Specificity: Mab1 clone P18 (CD61, anti-GPIIIa) Mab2 clone PM6/13 (CD61,anti-GPIIIa)

Source: Clone P18 (mAb IgG2a INSERM Clone PM6/13 (Mab IgG1) Cymbus

A sheep (Fab)'₂ anti-mouse total Ig—FITC reagent, DDAF, constitutes themost appropriate polyclonal reagent.

FIG. 3 is a saturation study on platelets for purified mAbs CD 61 P18and PM 6/13.

Protocol 3

1—Materials

a 7E3 monoclonal antibody sold under the tradename REOPRO® 2 mg/mldilutions in PBS BSA 0.1%, 3-fold from 200 μg/ml to 0.09 μg/ml

mAb P18: Ascite 1/800

mAb PM6/13: 10 μg/ml

DDAF

PRP

2—Method

{fraction (1/10)} dilution of each preparation of a 7E3 monoclonalantibody sold under the tradename REOPRO® in PRP. Incubation 5 min RT

Washing (in PBS BSA 0.1% 3000 rpm 10 min)

Pellet+PBS BSA 0.1% (vol equal to the initial volume of PRP)

20 μl of platelet preparation+20 μl of mAb P18, PM6/13 and IgG, negativecontrol.

Incubation 10 min RT

+100 μl DDAF 1/100 on each immunolabelling and on 40 μl of calibrationseries of the CYTOQUANT GP type. Incubation 10 min RT

+2 ml PBS BSA 0.1%→FCM.

EXAMPLE 2

Equivalent determinations were carried out using:

PM6/13 as Mab2, and

P18 as Mab1

in an in vitro model on whole blood with, as anti-aggregation agent, a7E3 monoclonal antibody sold under the tradename REOPRO® at variousconcentrations.

The results are assembled in FIG. 4.

EXAMPLE 3

Equivalent assays were carried out using:

P18 as Mab2

PM6/13 as Mab1

in an in vitro model using, as anti-aggregation agent, the RGD peptideat various concentrations.

The results are assembled in FIG. 5.

In Examples 2 and 3, the drug used (a 7E3 monoclonal antibody sold underthe tradename REOPRO® or RGD peptide) is diluted in a phosphate bufferin order to obtain a series of dilutions from 1 to 50 μg/ml. 5 μl aremixed with 45 μl of whole blood ({fraction (1/10)} dilution). Theincubation is carried out at room temperature for 30′ and then 150 μl ofbuffer are added.

To aliquots of 20 μl of diluted whole blood are added 20 μl of mAbtested at 10 or 20 μg/ml.

After incubation and addition of FITC-conjugated anti-mouse Ig Ab, acytometric analysis is carried out.

The number of mAbs molecules bound by platelets is calculated using acalibration straight line plotted using latex beads, in accordance withthe IQIF method.

EXAMPLE 4

Tests were carried out in vivo on patients treated with ReoPro.

In this case, a 7E3 monoclonal antibody sold under the tradename REOPRO®is not added. Whole blood is collected (20 μl diluted ¼ in physiologicalbuffer).

20 μl of mAb (P18 or PM 6/13) at 10 μg/ml are added. The incubation iscarried out for 10′ at room temperature (RT). 20 μl of reagent are thenadded and a cytometric reading is carried out after incubation (10′ RT)and appropriate dilution.

FIG. 6 shows the number of free receptors after the injection relativeto the total number of receptors. After 48 hours, the number of freereceptors is still less than 50%.

The mAbs used are PM6/13 for the determination of the total receptorsand P18 for the determination of the free receptors.

Claims:
 1. A method for determining the occupation of cellularGPIIb/IIIa receptors by a platelet anti-aggregating agent, comprisingthe steps of (a) determining the number of occupied receptors with afirst monoclonal antibody (“MAb1”) which is a competitor for said agentby determining the number of MAb1 bound to the receptors; (b)determining the number of total receptors with a second monoclonalantibody (“MAb2”) which is a non-competitor for said agent bydetermining the number of MAb2 bound to the receptors; and (c) deducingthe rate of occupation of the receptors by dividing the number of MAb1bound with the number of MAb2 bound.
 2. Method according to claim 1,characterized in that the number of receptors is measured by comparisonwith calibration curves.
 3. Method according to claim 1, characterizedin that the antibodies are directed against GPIIIa.
 4. Method accordingto claim 1, characterized in that the determination is carried out bylabeling the antibodies with the aid of a labeled antibody.
 5. Methodaccording to claim 4, characterized in that the antibody is labeled witha fluorochrome.
 6. Method according to claim 5, characterized in thatthe determination is carried out by quantitative cytometry.
 7. Methodaccording to claim 6, characterized in that the determination is carriedout by the indirect quantitative immunofluorescence method.
 8. Methodaccording to claim 1, wherein the Mab1 is the monoclonal antibodyPM6/13, the Mab2 is the monoclonal antibody SZ21 and the agent is an RGDpeptide.
 9. Method according to claim 1, wherein the Mab1 is themonoclonal antibody P2, the Mab2 is the monoclonal antibody PM6/13 andthe agent is the antibody 7E3 or fragments thereof.
 10. Method accordingto claim 1, characterized in that the sample to be tested is a bloodsample.
 11. Method according to claim 10, characterized in that thesample to be tested is a sample enriched in platelets.
 12. The method asset forth in claim 1, wherein said agent is a fibrinogen antagonist. 13.Assay kit intended for carrying out the method according to claim 1,characterized in, that it comprises at least: one monoclonal antibodyfor the GPIIb/IIIa receptor which is a competitor for a plateletanti-aggregating agent, and one monoclonal antibody specific for theoccupied or unoccupied GPIIb/IIIa receptor.
 14. The kit of claim 13,wherein the agent is a Fibrinogen antagonist.